2297

Journal of Food Protection, Vol. 70, No. 10, 2007, Pages 2297-2305 Copyright ©, International Association for Food Protection

Effect of Potassium Lactate and a Potassium Lactate-Sodium Diacetate Blend on Listeria monocytogenes Growth in Modified Atmosphere Packaged Sliced Ham

L. A. MELLEFONT* AND T. ROSS

Australian Food Safety Centre of Excellence, Tasmanian Institute of Agricultural Research, School of Agricultural Science, University of Tasmania, Private Bag 54, Hobart 7001, Tasmania, Australia

MS 07-051: Received 30 January 2007/Accepted 9 May 2007

ABSTRACT

Two commercially available organic acid salts, potassium lactate (PURASAL HiPure P) and a potassium lactate-sodium diacetate blend (PURASAL Opti.Form PD 4), were assessed as potential inhibitors of Listeria monocytogenes growth in modified atmosphere packaged (MAP) sliced ham in challenge studies. The influence of the initial inoculation level of L. monocytogenes (101 or 103 CPU g-I) and storage temperature (4 or g0C) was also examined. The addition of either organic acid salt to MAP sliced ham strongly inhibited the growth of L. monocytogenes during the normal shelf life of the product under ideal refrigeration conditions (4°C) and even under abusive temperature conditions (i.e., g0C). During the challenge studies and in the absence of either organic acid salt, L. monocytogenes numbers increased by l,OOO-fold after 20 days at goC and IO-fold after 42 days at 4°C. Both organic acid salt treatments were found to be listeriostatic rather than listericidal. The addition of either organic acid salt to the MAP ham also reduced the growth of indigenous microflora, i.e., aerobic microflora and lactic acid bacteria. The influence of these compounds on the risk of listeriosis in relation to product shelf life is discussed.

Unlike most foodbome pathogens, Listeria monocyto­ modify or treat long shelf life refrigerated products in such genes can grow, albeit slowly, at refrigeration temperatures, a way that the growth of L. monocytogenes is prevented, at in the presence of >5% salt, and in the absence of oxygen least within the normal shelf life of the product. Organic (37). Processed meats, including ham and pate, are often acids and their salts, applied singly and in combination, packaged under vacuum or modified atmospheres and have been shown by many studies to prevent or greatly stored under refrigeration to extend their shelf life, typically retard the growth of L. monocytogenes in processed meats being 6 to 8 weeks or more. Although initial contamination under both recommended temperatures of storage (at or be­ with L. monocytogenes, when present, is usually low, i.e., low 4°C) and mild temperature abuse (e.g., up to 10°C). <10 CPU g-l (15, 28), its potential for growth in ready­ Generally, studies have focused on the activity of the so­ to-eat meats that are eaten without further cooking prior to dium salts (2-4, 9, 14, 17, 18,21,22,27,29,31, 34, 40), consumption is, prima facie, considerable. Moreover, it is but several also consider potassium lactate or potassium well documented that L. monocytogenes can grow in many lactate in combination with sodium diacetate (19, 26, 32, such products during refrigerated storage, and such foods 33). Most studies have considered the activity of organic have caused, or been strongly implicated in, outbreaks of acid salts in sausage products (e.g., bratwurst, frankfurters, foodbome listeriosis that have resulted in fatalities (6-8, 23, saveloys), but others have studied their effectiveness in 25). cooked ham (4, 34). Mbandi and Shelef (21) considered the L. monocytogenes present on raw ingredients can be effectiveness in sterile uncooked comminuted beef emul­ killed reliably by the heat treatments routinely applied dur­ sion. ing smallgoods processing. During slicing and packaging There are a range of commercial products available after processing, however, recontamination can occur (16, based on the salts of organic acids that are intended to pro­ 18, 24, 30, 39). During the past decade, the food industry vide protection against the growth of undesirable microor­ has implemented hygiene practices and technologies that ganisms on foods. This project was undertaken to assess have greatly reduced the prevalence of L. monocytogenes the antilisterial efficacy of two such products on an Aus­ tralian smallgoods product, specifically, PURASAL HiPure on ready-to-eat foods (20, 28). Nevertheless, the ecology P (potassium lactate) and PURASAL Opti.Form PD 4 (a of the organism, particularly its ability to colonize food potassium lactate-sodium diacetate blend). The influence, processing plants (38), still results in L. monocytogenes be­ and possible interaction, of the presence of either antilis­ ing detected, typically, in -1 to 5%, or more, of processed terial product, the initial level of L. monocytogenes, and the meat products (1, 5, 15, 20, 28, 41). temperature of storage on the development of populations One approach to minimize the risk of listeriosis is to of L. monocytogenes in modified atmosphere packaged * Author for correspondence. Tel: + 61 (0)3 62 266278. Fax: + 61 (0)3 (MAP) sliced ham are investigated. The influence of indig­ 62 267450; E-mail: lyndal.mel1efo~utas.edu.au. enous microbiota is also considered. 2298 MELLEFONT AND ROSS ~ J. Food Prot., Vol. 70, No. 10

MATERIALS AND METHODS aging. Uninoculated control samples were prepared by adding 0.1 ml of sterile pw. To minimize the potential for cross-contamina­ Ham preparation. Hams were prepared under commercial tion, these control samples were prepared prior to the samples conditions by a large Australian manufacturer. During processing, requiring inoculation with the L. monocytogenes. antilisterial treatments were applied to hams prior to cooking. The To preserve the integrity of the gas mixture, a self-adhesive treatments were as follows: the addition of 3% (wtJwt finished rubber septum was applied to each sample package, and the sy­ product) of PURASAL Opti.Form PD 4 (a blend consisting of ringe was inserted into the pack through the septum. An inoculum 54.5 to 57.5% potassium lactate and 3.7 to 4.3% sodium diacetate: volume of 0.1 ml was added to a comer of the base of the package Purac Asia Pacific Pte. Ltd., Singapore) or the addition of 3% (wtJ wt finished product) of PURASAL HiPure P (58 to 62% potas­ that did not contain any ham. The package was then inverted and sium lactate). Both products were provided to the smallgoods pro­ gently shaken (by hand) so that the slices of ham moved across ducer by Fibrisol Service Australia Pty. Ltd. (Heatherton, Victoria, the inner surfaces of the container and each other. This was done Australia). Finished hams were sliced by the manufacturer and to maximize the likelihood of even distribution of cells of L. packaged in -50-g lots into commercial thermoformed ridge bot­ monocytogenes or sterile diluent throughout the sample. Imme­ tom packs, composed of polyethylene teraphthalate and polyeth­ diately after inoculation (or addition of sterile PW), the septum ylene, and produced on horizontal form fill. The packages were was covered with self-adhesive tape. Inoculated samples were ap­ propriately labeled and incubated at either 4 or 8°C in walk-in then sealed under MAP (30% CO2 and 70% N2) . Control and treated samples were transported to the laboratory by commercial cool-rooms. The duration of storage was :5.76 and :5.57 days for transport operators under refrigeration, but no temperature records samples stored at 4 and 8°C, respectively. were available. Sampling. At appropriate time intervals, three samples from Bacterial strains. Five strains of L. monocytogenes were each treatment or control were assessed for levels of L. monocy­ used in combination for all "inoculated" samples. L. monocyto­ togenes, concentrations of lactic acid bacteria, and aerobic plate genes Scott A (type strain) and L5/22 (cold-smoked salmon iso­ count (APC). The entire sample from each package was asepti­ late) were obtained from the School of Agricultural Science, Uni­ cally removed and diluted I: I in PW and stomached for 2 min. versity of Tasmania. Strains 20425, 20432, and 20423, all envi­ Further W-fold dilutions in PW were prepared as required, and ronmental isolates from a smallgoods factory, were supplied by 100- or 250-111 aliquots were surface plated with a spiral plater Silliker Microtech Pty., Ltd., Melbourne, Victoria, Australia. (Autoplate 4000, Spiral Biotech Inc., Bethesda, Md.) onto agar Strains were maintained on bead suspensions in nutrient broth plates as follows. PALCAM plates (Oxoid CM877, and SRl50 (Oxoid CMl, Oxoid Ltd., Hampshire, UK) with 15% glycerol and antibiotic supplement) for L. monocytogenes were incubated aer­ stored at -70°C. obically at 37°C for 48 h. The deMan Rogosa Sharpe agar (Oxoid Preparation of inocula. Before each experiment, isolates CM361) plates for lactic acid bacteria were incubated aerobically were resuscitated from cryogenic storage by plating onto brain at 25°C for 72 h. Plate count agar plates (Oxoid CM463 APHA heart infusion agar (Oxoid CM225) and incubating at 25°C for 48 Standard Plate Count Agar) for APCs were incubated at 20°C for h. A primary culture of each strain was prepared by touching a 72 h. Colonies were counted manually, and log(viable cell count) sterile loop to five colonies and inoculating 10 ml of prewarmed of the triplicate samples was averaged (±standard deviation) and (25°C) tryptone soya broth (Oxoid CM129) containing 0.6% yeast plotted against time. The maximum sample volume plated was extract (TSBYE; Oxoid L21). The primary culture for each strain 250 111 of the I: 1 dilution on quadruplicate plates. This permitted was incubated without shaking at 37°C for 24 h. The primary a maximum test sensitivity of 2 CFU g-I. cultures were then serially diluted in 0.1% peptone water (PW; Water activity (Aqualab CX2, Decagon Devices, Pullman, Oxoid Bacteriological Peptone L37 containing 0.85% NaCl) and Wash.) and direct measurement of the pH (pH meter 250A. Orion added to 50 ml of TSBYE broths in 125-ml side-arm flasks to Research Inc., Boston, Mass.) of the ham were also determined achieve a level of approximately 106 CFU ml". The cultures were from triplicate samples taken near the commencement of incu­ then incubated with shaking (80 oscillations min-I) in a water bation and periodically throughout the trial. bath (Ratek Instruments Pty. Ltd., Victoria, Australia) at woe. Growth was monitored turbidimetrically (540 nm) until percent RESULTS transmittance had decreased to -20%. These steps were under­ taken to generate exponentially growing inocula of -108 CFU Because of the time taken to transport the hams from ml"! and acclimated to the chill temperatures considered repre­ the commercial processor to the laboratory and to prepare sentative of a smallgoods processing facility. the inoculum, the time between preparation of the hams and One-milliliter aliquots of the 10°C broth cultures of each of commencement of the challenge trial was 11 days. Changes the five strains were added to a sterile 30-ml bottle and vortexed in the microbial ecology and physicochemical attributes of for I min. This "cocktail" suspension was then diluted in PW, the product were expected to commence from the time of prechilled to WOC, to achieve levels of inocula such that final packaging. Thus, all incubation times reported below relate levels in inoculated samples were - 101 or - 103 L. monocyto­ genes CFU g-I ham. The amount of L. monocytogenes in the to the time since commercial preparation, not the time of cocktail suspension was determined by viable count. commencement of the challenge study. The manufacturer's shelf life recommendation for the untreated commercial Inoculation of ham. Upon receipt at the University of Tas­ product under the recommended refrigerated storage con­ mania laboratories, control (no treatment) and treated samples ditions was -42 days. were stored at 2°C for 2 days prior to inoculation with the L. monocytogenes cocktail and commencement of the challenge trial. The challenge trial was conducted once for each com­ Low- and high-density inocula, prepared as described above, bination of control or antilisterial product, incubation tem­ were aseptically injected onto the MAP sliced ham products (-50 perature, and level of L. monocytogenes, with triplicate g per package) with a hypodermic syringe to penetrate the pack­ samples analyzed at each sampling time. J. Food Prot., Vol. 70. No. 10 EFFECT OF ANTIMICROBIALS ON L. MONOCYTOGENES IN MAP HAM 2299

TABLE 1. pH changes over time in control and PURASAL-treated ham samples during storage at either 4 or 8°C (n = 3)a Sample time (days): Temp (0C) Ham treatment 21 38 46 60 74 88

4 Control (untreated, no 6.44 ± 0.03 6.40 ± 0.13 NT NT 6.15 ± 0.07 6.48 ± 0.06 L. monocytogenes) Untreated, ~101 CFU g-I 6.46 ± 0.02 6.44 ± 0.04 6.42 ± 0 6.19 ± 0.04 6.07 ± 0.03 6.43 ± 0.10 L. monocytogenes Untreated, -103 CFU g-I 6.40 ± 0.12 6.50 ± 0.03 6.44 ± 0.05 6.20 ± 0.05 6.10 ± 0.003 6.41 ± 0.11 L. monocytogenes Hi Pure P, ~ 101 CFU g-I 6.40 ± 0.01 6.44 ± 0.02 6.48 ± 0.06 6.12 ± 0.05 6.19 ± 0.03 6.20 ± 0.04 L. monocytogenes Hi Pure P, ~ 103 CFU s' 6.48 ± 0.05 6.44 ± 0.03 6.47 ± 0.08 6.16 ± 0.03 6.23 ± 0.04 6.21 ± 0.03 L. monocytogenes Opti.Form PD 4, 101 CFU 6.47 ± 0.04 6.44 ± 0.03 6.45 ± 0.06 6.08 ± 0.10 6.24 ± 0.04 6.21 ± 0.03 g-I L. monocytogenes Opti.Form PD 4, ~ 103 CFU 6.44 ± 0.05 6.45 ± 0.05 6.43 ± 0.04 6.19 ± 0.08 6.23 ± 0.04 6.22 ± 0.01 s' L. monocytogenes Sample time (days): Temp COC) Ham treatment 13 20 24 31 41 61 68

8 Control (untreated, no 6.23 ± 0.05 6.14 ± 0.02 6.52 ± 0.06 6.20 ± 0.01 NT 5.85 ± 0.11 5.59 ± 0.24 L. monocytogenes) Untreated, ~101 CFU g-I 6.21 ± 0.04 6.16 ± 0.04 6.45 ± 0.06 6.17 ± 0.04 6.37 ± 0.20 5.95 ± 0.10 5.71 ± 0.14 L. monocytogenes Untreated, ~103 CFU s' 6.19 ± 0.02 6.18 ± 0.02 6.45 ± 0.03 6.20 ± 0.02 6.27 ± 0.21 5.84 ± 0.12 5.75 ± 0.17 L. monocytogenes Hi Pure P, -101 CFU g-I 6.24 ± 0.03 6.17 ± om 6.45 ± 0.04 6.22 ± 0.01 6.49 ± 0.01 6.11 ± 0.02 6.22 ± 0.03 L. monocytognes Hi Pure P, ~ 103 CFU g-l 6.23 ± 0.04 6.16 ± 0.04 6.46 ± 0.03 6.14 ± 0.02 6.44 ± 0.01 6.23 ± 0.09 6.17 ± 0.03 L. monocytogenes Opti.Form PD 4, -101 CFU 6.21 ± 0.05 6.18 ± 0.03 6.45 ± 0.03 6.19 ± 0.02 6.53 ± 0.10 6.15 ± 0.07 6.20 ± 0.03 s' L. monocytogenes Opti.Form PD 4, -103 CFU 6.25 ± 0.06 6.19 ± 0.03 6.43 ± 0.02 6.16 ± 0.04 6.46 ± 0.06 6.22 ± 0.08 6.16 ± 0.03 g-I L. monocytogenes a NT, not tested.

Water activity, gas mixture, and pH changes. The untreated hams, both uninoculated and inoculated with L. water activity of control and treated ham samples showed monocytogenes, the pH declined after 40 days to -5.6 to little change during storage at either 4 or 8°C (data not 5.7. shown). Moreover, the addition of either a sterile diluent or The gas composition of selected samples, assessed pe­ small volume of inoculum to the hams did not affect water riodically throughout the trial, showed no systematic chang­ activity. Overall, untreated hams had a higher average water es (data not shown). activity (0.969 ± 0.003; n = 27) than hams treated with either the potassium lactate-sodium diacetate blend (0.959 Untreated control ham. The data for the uninoculated ± 0.005; n = 18) or potassium lactate (0.959 ± 0.002; n and inoculated untreated hams provide the baseline against = 18). which the efficacy of the two treatments can be assessed. The pH values of the control (untreated) and treated The APC reached maximum population density by day 54 ham samples over time of incubation at 4 and 8°C are at 4°C and day 34 at 8°C for both uninoculated and un­ shown in Table 1. The pattern of response was initially treated hams inoculated with either low or high numbers of similar for all ham types at 4°C. The pH remained constant L. monocytogenes (Figs. la through lc and 2a through 2c). at -6.4 up to 46 days postprocessing and then declined to In all cases, the APC mainly consisted of lactic acid bac­ -6.2. Thereafter, the pH of treated hams remained un­ teria. L. monocytogenes was not recovered from uninocu­ changed. Untreated hams, both inoculated and uninoculat­ lated control samples stored at either 4 or 8°C (Figs. la and ed, returned to a pH similar to that at commencement of 2a) with a level of detection of <2 CFU g". the trial. At 8°C, pH changes were similar for all hams up L. monocytogenes inoculated into untreated samples to 31 days postprocessing, with pH fluctuating between and stored at 4 or 8°C showed growth. At 4°C, approxi­ -6.2 and 6.4. This pattern continued for the duration of mately 1 log of growth was observed 42 days after inoc­ the trial for hams treated wit~either antilisterial agent. For ulation for ham with either low (Fig. Ib) or high (Fig. Ic) 2300 MELLEFONT AND ROSS ~ 1. Food Prot., Vol. 70, No. 10

a a

T ~ __ -m----8--~ /~-t=S--1i--!I" _e----ijI ~f t--_'L---~- ~~...... ""!--..-~---+ _ .. t~~ 1: 1­ /Ai"- ­ Gf'I " :- 1­ I~L Jr

~ • • o 10 20 30 40 50 60 70 80 90 o 10 20 30 40 50 60 70 80 90 Time (days) Time (days) b b

_t-tj:::::===t~t:==~ -~~ , i f 1 IF'­

I r------r --, • • • o 10 20 30 40 50 60 70 80 90 o 10 20 30 40 50 60 70 80 90 Time (days) Time (days) C c 10.0 Ti'------, 10.0 Ti------, 9.0 9.0 8.0 8.0 T _.J:~- 7.0 -e- I" 1 ..-f---BJ 7.0 ,$-1...... -w--!'"- -- ~ 6.0 6.0 / - -+ 5.0 5.0 ,tj~ 1 ­ 4.0 4.0 -./...----_ ,-- t t --1 3.0 3.0 2.0 2.0 1.0

1.0 0.0 ~ I I I I I I

0.0 ·1 I I I I I I o 10 20 30 40 50 60 70 80 90 o 10 20 30 40 50 60 70 80 90 Time (days) Time (days) FIGURE 2. Growth of aerobic microflora (- - 0 - -), lactic acid FIGURE 1. Growth of aerobic microflora (- - 0 - -), lactic acid bacteria (- - (> - -), and L. monocytogenes (e) in untreated mod­ bacteria (- - (> - -), and L. monocytogenes (e) in untreated mod­ ified atmosphere packaged sliced ham stored at 8°C where (a) ified atmosphere packaged sliced ham stored at 4°C where (a) uninoculated control, (b) inoculated with low numbers (-101 uninoculated control, (b) inoculated with low numbers (-101 CFU g-I) of L. monocytogenes, and (c) inoculated with high CFU s:') of L. monocytogenes, and (c) inoculated with high numbers (-]()3 CFU s:') of L. monocytogenes. Standard devi­ numbers (-]()3 CFU s:') ofL. monocytogenes. Standard devi­ ation (n = 3) is also plotted. For uninoculated control samples, ation (n = 3) is also plotted. For uninoculated control samples, data plotted represent a determination of <2 CFU e:'. data plotted represent a determination of <2 CFU s'. goe, -2 days. In all inoculated and untreated hams, the growth of L. monocytogenes in the product appeared to pla­ added numbers of L. monocytogenes. More growth was ob­ teau when the total number of viable aerobic bacterial cells served, - 3 log, at goe after 20 days (postinoculation) and in the samples reached -108 eFU g-l. was also independent of inoculum size (Fig. 2b and 2c). Initiation of growth at 4°e appeared to be considerably de­ Treated ham. Figures 3a and 3b and 4a and 4b show layed, -20 days postinoculation, compared with that at the effect of the addition of the potassium lactate-sodium J. Food Prot., Vol. 70, No. 10 EFFECT OF ANTIMICROBIALS ON L. MONOCITOGENES IN MAP HAM 2301

a a 10.0 10.0 9.0 9.0 8.0 8.0 T 7.0 7.0 T I 6.0 6.0 A 5.0 5.0 " ~~/ ­ .fJ.,.. ok 4.0 4.0 ~/ 3.0 3.0 2.0 2.0 I~ 1.0 1.0 0.0 0.0 0 10 20 30 40 50 60 70 80 90 0 10 20 30 40 50 60 70 80 90 Time (days) Time (days)

b b 10.0 10.0 9.0 9.0 8.0 8.0 7.0 7.0 "T'" 6.0 6.0 5.0 5.0 T 4.0 1, 4.0 3.0 ~. ~ 3.0 • I,•C:t-- .<,t,>I I :1 2.0 \1-'-'1 2.0 ~ \ " 1. , I II 1.0 1.0

I ~_-.-_--,-_-, ~_~_~ ~----J 'f---i 1 0.0 __ 0.0 "-4 0 10 20 30 40 50 60 70 80 90 o 10 20 30 40 50 60 70 80 90 Time (days) Time (days) FIGURE 3. Growth of aerobic microfiora (- - 0 - -), lactic acid FIGURE 4. Growth of aerobic microflora (- - 0 - -), lactic acid bacteria (- - <> - -), and L. monocytogenes (e) at 4°C in modified bacteria (- - <> - -), and L. monocytogenes (e) at 8°C in modified atmosphere packaged sliced ham treated with potassium lactate­ atmosphere packaged sliced ham treated with potassium lactate­ sodium diacetate (PURASAL Opti.Form PD 4) and inoculated sodium diacetate (PURASAL Opti.Form PD 4) and inoculated with either (a) low numbers (-101 CFU s:')or (b) high numbers with either (a) low numbers (-101 CFU g-l) or (b) high numbers (-]()3 CFU s:') ofL. monocytogenes. Standard deviation (n = (-]()3 CFU s:') ofL. monocytogenes. Standard deviation (n = 3) is also plotted. 3) is also plotted. diacetate blend, and Figures 5a and 5b and 6a and 6b show Both treatments at both temperatures also suppressed the effect of potassium lactate on the changes in microbial the growth of indigenous bacteria in terms of growth rate populations, including added L. monocytogenes, in MAP and time of initiation of growth compared with untreated sliced ham. In all cases, it was apparent that the treatments hams. It is important to note that the APC includes the greatly reduced the extent of microbial growth when com­ introduced L. monocytogenes so that, in some cases, the pared with untreated hams (Figs. lb and lc and 2b and 2c). growth of indigenous bacteria that are present at concen­ Regardless of inoculum level, the growth of L. mono­ trations less than ~ 103 CFU g-I can be masked by the cytogenes was prevented for 76 days (postinoculation) at nongrowing L. monocytogenes population in the high in­ 4°C in both potassium lactate-sodium diacetate (Fig. 3a and oculum treatments. 3b) and potassium lactate (Fig. 5a and 5b) treated hams. For hams treated with potassium lactate-sodium diac­ Similarly, no growth of L. monocytogenes was observed on etate, the APC remained unchanged for 55 days (postin­ potassium lactate-treated hams after 57 days (postinocula­ oculation) at 4°C, regardless of L. monocytogenes inoculum tion) at 8°C (Fig. 6a and 6b). A small increase (-0.5 log level (Fig. 3a and 3b). Thereafter, numbers increased slow­ CFU g-I) in L. monocytogenes numbers was apparent in ly, in comparison to control hams, to ~5 and 4.5 log CFU the last samples taken, i.e., 57 days postinoculation, for g-I in low and high inoculated hams, respectively. At 8°C, potassium lactate-sodium diacetate treated hams stored at APC growth was suppressed for 20 days postinoculation. 8°C. With the Student's t test, the difference between the Thereafter, the APC increased to -6 log CFU g-I at a mean of those counts and the mean of the preceding counts slower rate than observed in control hams inoculated with was found to be significant (P < 0.05) for the samples with L. monocytogenes (Fig. 2b and 2c). low L. monocytogenes inoculum (Fig. 4a) and highly sig­ For hams treated with potassium lactate, the APC re­ nificant (P < 0.001) for the samples with high L. mono­ mained relatively unchanged during storage at 4°C, i.e., for cytogenes inoculum (Fig. 4b)~ 76 days postinoculation (Fig. 5a and 5b). A small increase 2302 MELLEFONT AND ROSS -... J. Food Prot., Vol. 70, No. 10

a a

10 20 o 10 20 30 40 50 60 70 80 90 o 30 40 50 60 70 80 90 Time (days) Time (days)

b b

\ I¥'- ...... r ~-../ L ~ t ...... _~---~ o 10 20 30 40 50 60 70 80 90 o 10 20 30 40 50 60 70 80 90 Time (days) Time (days) FlGURE 5. Growth of aerobic microjlora (- - D - -), lactic acid FIGURE 6. Growth of aerobic microjlora (- - D - -), lactic acid bacteria (- - 0 - -), and L. monocytogenes (e) at SoC in modified bacteria (- - 0 - -), and L. monocytogenes (e) at 4°C in modified atmosphere packaged sliced ham treated with potassium lactate atmosphere packaged sliced ham treated with potassium lactate (PURASAL HiPure P) and inoculated with either (a) low numbers (PURASAL HiPure P) and inoculated with either (a) low numbers (_]01 CFU g-I) or (b) high numbers (-]()3 CFU s') of L. (~]OI CFU s') or (b) high numbers (~]()3 CFU s') of L. monocytogenes. Standard deviation (n = 3) is also plotted. monocytogenes. Standard deviation (n = 3) is also plotted.

DISCUSSION in numbers may have occurred in the hams inoculated with The five L. monocytogenes strains used in these ex­ low numbers of L. monocytogenes (Fig. Sa); however, the periments were grown to late exponential phase and accli­ response fluctuated over the duration of storage. Note that mated to growth at chill temperature (I O°e) to minimize the APC mainly consists of L. monocytogenes in the high the lag time on introduction to the ham environment. This inoculated hams (Fig. Sb), thus masking the response of approach appears to have been successful as, in the untreat­ indigenous microflora. As for potassium lactate-sodium di­ ed and inoculated controls (Fig. 2b and 2c) at 8°C, there is acetate treated hams at 8°C (Fig. 4a and 4b), the APC is little evidence of a lag phase before the commencement of suppressed for the first 20 days of storage postinoculation. growth. At 4°C, however, there is a lag time of -20 days Thereafter, the APC increases. If the APC data for all treat­ (Fig. Ia and Ib). The use of a mixture of five strains of L. ed and inoculated hams are plotted together, it is apparent monocytogenes was intended to minimize the possibility that the rate of increase is slower in potassium lactate-treat­ that any observed inhibition of growth observed would be ed hams than in those treated with the potassium lactate­ strain-speci fico sodium diacetate blend (data not shown). To more clearly illustrate the effects of the two treat­ The response of indigenous lactic acid bacteria in the ments on the potential for L. monocytogenes growth in treated and inoculated hams was similar to that of the APC, MAP stored ham, Figure 7a and 7b presents a direct com­ particularly when growth was observed. However, when the parison of the population growth of L. monocytogenes in APC remained static, it was apparent that the numbers re­ untreated controls, and the respective treatments, at each of covered on deMan Rogosa Sharpe varied between sampling the storage temperatures and L. monocytogenes inoculum times. For example see Figure 3b for hams treated with levels studied. The results support the reported effective­ potassium lactate-sodium diacetate, inoculated with high ness of both potassium lactate and combined potassium lac­ numbers of L. monocytogenes, and stored at 4°C. tate and sodium diacetate under both recommended storage J. Food Prot., Vol. 70, No. 10 EFFECT OF ANTIMICROBIALS ON 1. MONOCITOGENES IN MAP HAM 2303

a 5.0 ,------=0 ates an additional hurdle to the growth of organisms present in lower numbers. This phenomenon has been termed the t Jameson Effect (10, 11, 13, 35). Expressed simply, the Ja­ 4.0 ! f f ! t f meson Effect describes the often-observed phenomenon I ~ I that when one microbial species in an environment reaches 3.0 II i' • i , • stationary phase, all other species present also enter sta­ ! t tionary phase, irrespective of whether they are at levels nor­ 2.0 I f mally associated with their maximum population density. • f The phenomenon is not yet fully explained but can account 1.0 i , , ,I " ,! j for the observations summarized in Figure 7a and 7b in ! ! which the maximum population density achieved by intro­ 0.0 'I duced L. monocytogenes appears to be dependent on their 10 20 30 40 50 60 70 80 90 Time (days) initial density in the challenge trial. We interpret this as occurring because the concentration of other bacteria ini­ b tially present on the ham was the same, irrespective of the 8.0 level of introduced L. monocytogenes, and because those 7.0 bacteria reached stationary phase at the same time during 6.0 f f the respective challenge trial conditions (i.e., 4 or SoC). The I growth of L. monocytogenes was suppressed after the same 5.0 I amount of time. Consequently, the final levels reached by 4.0 , t I L. monocytogenes in the product are dependent on the lev­ 3.0 i , iii • els initially introduced. Figure 2b and 2c illustrates the ef­ 2.0 fect most clearly by showing that the cessation of the growth of L. monocytogenes corresponds closely with the 1.0 , , , onset of stationary phase of the aerobic microflora, which 00 is dominated by the lactic acid bacteria. This effect was 10 20 30 40 50 60 70 also noted by Stekelenburg (33). Glass et al. (14) also com­ Time (days) mented on the contribution of lactic acid bacteria to sup­ FIGURE 7. Growth at (a) 4°C and (b) BOC of either high (circles) pression of pathogen growth in long shelf life processed or low (squares) levels ofL. monocytogenes inoculated into ham meats and stated that "manufacturers must optimise the treated with either potassium lactate-sodium diacetate (PURAS­ critical balance between increasing shelf life and permitting AL P Opti.Form PD 4; open symbols) or potassium lactate (PUR­ the growth of spoilage lactic acid bacteria that compete ASAL HiPure P; closed symbols) compared with untreated control L. monocytogenes samples (shaded symbols). Standard deviation (n = 3) is also with for nutrients." plotted. Although full characterization of the likely reduction in risk of listeriosis due to use of listeriostatic agents such as organic acid salts would require extensive modeling, conditions (4°C) and inappropriate storage conditions (SOC) general trends can be inferred from the results in this study. for MAP ham. Although growth was clearly possible in the Given the low number of L. monocytogenes typically pres­ untreated ham product, the addition of the potassium lac­ ent on processed meats at the point of manufacture, the tate-sodium diacetate treatment almost completely elimi­ delay in growth induced by the presence of organic acid nated the growth of L. monocytogenes at 4 or SoC (Figs. salts (and CO2) , in combination with the growth-suppress­ 3a and 3b and 4a and 4b), while hams treated with potas­ ing effects of high concentrations of lactic acid bacteria or sium lactate completely suppressed the growth of L. mono­ other indigenous bacteria, would be expected to reduce the cytogenes at 4°C for 90 days (Fig. 5a and 5b) and for 60 risk of listeriosis from vacuum-packed or MAP processed days at SoC (Fig. 6a and 6b). Consistent with other reports meats. This is expected because, once any L. monocyto­ (3,4,14, 18,21,22,26,29,32-34), neither treatment was genes present was able to grow, other bacteria might have listericidal at the levels used but was listeriostatic. Samelis reached concentrations high enough to suppress L. mono­ et al. (29), however, found that 6% lactate was listericidal cytogenes growth, i.e., by the Jameson Effect. The magni­ in some processed meat products. tude of this suppression might also depend on the relative An additional level of protection against the growth of concentrations of lactic acid bacteria and L. monocytogenes L. monocytogenes in MAP or vacuum-packed products may initially present. For this reason, it is important to undertake be afforded by the presence and growth of lactic acid bac­ studies with realistic initial levels of L. monocytogenes teria. Lactic acid bacteria have an ecological advantage in (e.g., ::;10 CFU g-l), as noted by Porto et al. (26) and the such products and usually become numerically dominant present study. during storage (36) but without necessarily causing spoilage The beneficial effect of the addition of organic acid of the product. Homofermentative lactic acid bacteria, in salts in reducing risk from L. monocytogenes could be less­ particular, can reach high concentrations in vacuum-packed ened, however, if the growth suppression applied equally to or MAP products but without causing overt spoilage (12). indigenous bacteria and product shelf lives were extended The presence of high concentlttions of other bacteria ere­ as a result. Inhibition of the growth of lactic acid bacteria 2304 MELLEFONT AND ROSS ~ J. Food Prot., Vol. 70. No. 10 and APC was seen in this study (Figs. 3 through 6) and spoilage bacteria and pathogenic contaminants. This sug­ potentially enables an extension of product shelf life. Sa­ gests that the potential for shelf life extension achievable melis et al. (29) noted less pH reduction during prolonged with additions of organic acid salts must be considered to­ storage of untreated controls than with organic acid salt gether with the potential for L. monocytogenes contamina­ treated pork frankfurters. Similar results were observed in tion and growth in the product. Further studies to charac­ this study for ham stored at goC (Table 1). This is possibly terize these interactions are being undertaken. because of the inhibition of lactic acid bacteria growth and delay of spoilage processes. Stekelenburg (33) reported in­ ACKNOWLEDGMENTS hibition of the growth of lactic acid bacteria and resultant This study was funded by Purac Asia Pacific Pte. Ltd. and Fibrisol extension of shelf life of 75 to 125%, and in an earlier Service Australia Pty. Ltd., and we sincerely thank Mr. Michael Wain­ study, Stekelenburg and Kant-Muermans (34) reported that wright for his coordination of the project. PURASAL Opti.Form PD 4 the growth of L. monocytogenes was more inhibited than and PURASAL HiPure P were kindly supplied by Purac Asia Pacific Pte. Ltd. Hams were provided. and processed, by Don Smallgoods Co. Pty. the growth of a lactic acid bacterium, Lactobacillus cur­ Ltd. We also thank Ms. Fiona Birrell and Mrs. Lauri Parkinson for their vatus. Extension of shelf life to exploit the inhibition of expert technical assistance in undertaking the laborious microbiological growth of other microorganisms could lessen the antilister­ testing and Ms. Jill Anderson, Silliker Microtech Laboratories. Melbourne. ial benefits of organic acid salt treatments, because the for provision of the L. monocytogenes cultures. growth of L. monocytogenes, while delayed by the treat­ ment, might eventually be possible because of the extended REFERENCES shelf life of the product. Shelf lives of 75 to 90 days for I. Angelidis, A. S., and K. Koutsoumanis. 2006. 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